Myceliophthora thermophila (MtL) to a resolution of 1.62 Å together with a thorough structural comparison with other members of the CAZy family AA1_3 that comprises fungal laccases from ascomycetes. The recombinant protein produced in A. oryzae has a molecular mass of 75 kDa, a pI of 4.2 and carries 13.5 kDa N-linked glycans. In the crystal, MtL forms a dimer with the phenolic substrate binding pocket blocked, suggesting that the active form of the enzyme is monomeric. Overall, the MtL structure conforms with the canonical fold of fungal laccases as well as the features specific for the asco-laccases. However, the structural comparisons also reveal significant variations within this taxonomic subgroup. Notable differences in the T1-Cu active site topology and polar motifs imply molecular evolution to serve different functional roles. Very few surface residues are conserved and it is noticeable that they encompass residues that interact with the N-glycans and/or are located at domain interfaces. The N-glycosylation sites are surprisingly conserved among asco-laccases and in most cases the glycan displays extensive interactions with the protein. In particular, the glycans at Asn88 and Asn210 appear to have evolved as an integral part of the asco-laccase structure. An uneven distribution of the carbohydrates around the enzyme give unique properties to a distinct part of the surface of the asco-laccases which may have implication for laccase function–in particular towards large substrates.Laccases of different biological origins have been widely investigated and these studies have elucidated fundamentals of the generic catalytic mechanism. However, other features such as surface properties and residues located away from the catalytic centres may also have impact on enzyme function. Here we present the crystal structure of laccase from  Like other multicopper oxidases, laccases operate via catalytic Cu-centers and coupling of the oxidative reaction to reductive cleavage of atmospheric oxygen into water. Laccases are produced by fungi, plants and bacteria and among their .10.3.2 pwww.cazy.org; [Laccases have been classified as members of the AA1 enzyme family of auxiliary activities in the CAZY classification system . In thihttp://www.rcsb.org/) the structures of laccases have been thoroughly investigated, and were recently reviewed by Hakulinen & Rouvinen [With more than 100 depositions of laccases in the protein databank  [MaL from Melanocarpus albomyces [TaL from Thielavia arenaria [BaL from Botrytis aclada [AnL from Aspergillus niger [AnL display similar, but not identical, dimers that pack head-to-head with the T1-pockets from the two protomers facing each other. Whether this dimeric arrangement exists in solution and if it has any biological relevance remain an open issue [With the aim of elucidating the structure and function of asco-laccases, we have undertaken the structure determination of the asco-laccase from the thermophilic fungus la (MtL) , The strlbomyces , 24, TaLarenaria , BaL fros aclada , 27 and us niger  . The concentrated crude extract was thawn from -80°C in a water bath, centrifuged for 10 min at 10 000 x g to remove any particles, and then loaded onto a 1000 ml Sephadex G-75 column. The resin was packed in a XK50/60 column and pre-equilibrated with SEC-buffer . Fractions displaying laccase activity were pooled, concentrated and subjected to a second round of SEC. The SEC fractions with laccase activity (see 2.6) were pooled, concentrated and buffer exchanged to 20 mM Tris-acetate pH 8.0. Aliquots were stored at -80°C. The concentration of purified MtL samples was determined from A280nm and the molar extinction coefficient for MtL .MtL was characterized by denaturing SDS-PAGE run on a BioRad TGX 4–15% gradient gel and non-denaturing isoelectric focusing (IEF) using a NOVEX IEF gel pH 3–7, following the protocols given by the manufacturers. Protein bands were stained with Coomassie Brilliant Blue.MtL in solution was further probed by analytical size-exclusion chromatography on a Superdex 200 HR 10/30 column (Pharmacia). The column was equilibrated with 50 mM NaH2PO4, 150 mM NaCl, pH 7 and calibrated using a set of molecular weight protein standards (Bio-Rad) comprising bovine thyroglobulin (670 kDa), bovine gamma globulin (158 kDa), chicken ovalbumin (44 kDa) and horse myoglobulin (17 kDa). The MtL sample and the standards were all eluted at 0.4 ml/min.The homogeneity of MtL was measured by mass spectrometry using an Autoflex Speed MALDI-TOF Instrument (BRUKER) calibrated with Bovine Serum Albumin (BSA) as molecular weight standard . MtL was diluted in 0.1% tri-fluoro-acetic acid (TFA) to a final concentration of 20 μM, and mixed 1:1 with a saturated stock of α-cyano-4-hydroxycinnamic acid (α-CHCA) matrix in acetonitrile/TFA (33 v/v% / 0.1 v/v%).The mass of intact 3DCE instrument (Agilent Technologies). A 2.2 mg/ml solution of MtL was injected and eluted using 20 mM Na/K-phosphate as running buffer. Experiments were performed at pH 5 and pH 8, respectively. The capillary (0.05 mm in diameter and activated with 1 M NaOH for 20 minuttes) was kept at 15°C, and a constant voltage of 25 kV was applied.CE analysis was performed under native conditions with a HP-1 PEG 6000 was used for dilution of the enzyme [530 = 65 000 M-1 cm-1; [530nm min-1) within the linear range (0.1 to 0.4 AU).Laccase activity was measured using syringaldazine  as substrate. The assay was performed at room temperature with 0.28 mM SGZ in 25 mM Tris-maleate buffer pH 7.0, in a total volume of 4.4 ml. For stabilization, a buffer supplemented with 50 g Le enzyme . A HP 84-1 cm-1; ). The en-1. The high solubility of the protein is likely due to the glycosylation of the protein. Crystals formed under several conditions, but most of them diffracted poorly. Crystals obtained from condition C2  in the JSCG++2 screen from Jena Bioscience were further optimized in a manual setup using grid screens , additives and variation of drop ratios. All crystallization experiments were performed at room temperature, and both sitting and hanging drop vapor-diffusion setups were tested. Replacement of MgCl2 with CaCl2 improved the quality of the crystals. The crystal used for data collection was grown in a hanging drop setup with 3 μl protein (66 mg ml-1) and 2 μl reservoir solution equilibrated over a 500 μl reservoir . Attempts were made to soak the substrates 2,6-dimethoxy phenol  and 2,2’-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) into the crystals by adding solid substrate to the drop as described by Kallio et al. [Initial crystallization conditions were identified from screening with commercial kits: JCSG+ (Qiagen), PACT (Qiagen), Crystal Screen (Hampton Research) and Index HT (Hampton Research) using an Oryx 8 robot (Douglas Instruments) and a protein concentration of 100 mg mlXDS [SCALA [MOLREP [Melanocarpus albomyces laccase  as sear.; [COOT  and both[REFMAC5  and PHENX.REFINE  were usePROCHECK  was usedi.e. the four copper ions plus two calcium ions from the crystallization condition. The MtL structure is deposited in the Protein Data Bank (www.rcsb.org) with entry code 6F5K.Refinement statistics and model data are summarized in http://www.ebi.ac.uk/pdbe/prot_int/pistart.html [Structural alignments and graphics were made with PYMOL . The strart.html .MtL was produced by heterologous expression in A. oryzae. Highly pure and active laccase was obtained from the crude extract by repeated size exclusion chromatography. The purified enzyme displayed a specific activity of 65 U/mg on the synthetic phenolic laccase substrate syringaldazine at pH 7, which is in good agreement with the activity data reported by Berka et al. [MtL is a thermostable and heavily glycosylated protein, and it displays an activity half-life around 140 min at 65 oC   is directly involved in crystal packing mediating contacts between two symmetry related molecules as it coordinates to Asp530 and Asp534 in one molecule and the carbonyl oxygen of Val341 in another molecule. The coordination sphere is completed by four ligating water molecules. CA606 coordinates to the carbonyl oxygen of Leu363 and five water molecules and may help to stabilize the local loop conformation. The interactions mediated by CA605 could explain the positive effect on the crystal quality observed for Ca2+ ions, due to their more variable coordination geometry [The crystal structure of L  as searN-x-T/S) . In totaMtL consists of 559 amino-acid residues [MtL shares the overall architecture typical for 3-domain laccases [Mature residues . Only thlaccases . It consMtL, MaL, TaL, BaL and AnL) reveals that this fungal subgroup is rather diverse, in terms of sequence, structure and function. The asco-laccases belong to the orders of Sordariomycetes , Leotiomycetes (BaL) and Eurotiomycetes (AnL), respectively. At the sequence level, this is reflected in sequence identities ranging from 70–75% between laccases within the order of Sordariomycetes to 33–42% when comparing across the different orders . A third disulphide bridge (Cys4-Cys12) located in the N-terminal extension is conserved in all the structures, except for AnL which has a disordered N-terminal extension that could not be modelled in the crystal structure [BaL displays a fourth disulphide bridge between Cys201-Cys209 that seems to stabilize an extended loop  and the loops  near the putative, novel binding site described for ABTS [While the catalytic copper centres, including ten histidines and one cysteine that acts as Cu-ligands are strictly conserved, the comparison of the five asco-laccases with known structure (t orders . Accorditoms see . In contCα-atoms . BaL andtructure . BaL disloop see . Among MMtL numbering) and a cysteine (Cys503) in a trigonal planar arrangement; less than 4 Å from the Cu two non-polar residues are located in the axial positions. One of the axial ligands is invariably an Ile, while the nature of the other axial ligand varies. Oxidation of phenolic substrates occurs via electron transfer to the T1-Cu. The electrons are subsequently shuttled to the TNC-site where the reduction of molecular oxygen takes place [MaL in complex with the substrate 2,6-DMP [MtL numbering), assumed to be the primary electron acceptor. Another residue that is highly conserved in the T1-pocket of fungal laccases is Glu235 (either Glu or Asp). This negatively charged residue has been shown to play an important role in the oxidation of phenolic substrates, possibly by facilitating proton abstraction [The shape of the pocket defined by several loops (vide supra) differs significantly between the basi- and asco-laccases, but also the latter group of enzymes shows significant differences. The T1-Cu is coordinated by two histidines , medium potentials for asco-laccases  and low potentials for laccases from plants and bacteria  [MtL, MaL TaL, and BaL have a Leu in the second axial position, AnL resembles more the basi-laccases by having a Phe instead (Phe526). Based on the redox-potential-sequence-correlation described above, this could suggest that AnL has a higher redox potential. However, the redox potential determined for AnL is in the same range as for the other asco-laccases: AnL 0.45 V [MtL 0.47 V [MaL 0.48 V [TaL 0.51 V [BaL [The redox potential of the T1-Cu has been correlated with the nature of the second axial non-polar ligand . Typicalosition) , 53. MtLL 0.45 V ; MtL 0.4stead Phe6. Based L 0.51 V . On the tead Phe5. Based oMtL-MaL-TaL subdivision of asco-laccases , and in both BaL and AnL an additional polar residue (Glu497 in BaL) is found at the bottom of the pocket  than expected for a monomeric species  but could equally well be explained by a non-globular shape of the heavily glycosylated enzyme. Based on these data we conclude that MtL is functional in its monomeric form, which also is the predominant form in solution.All crystal structures of asco-laccases except for elevance , 26. AnLaL dimer . The res channel . It shouprotomer . Combinesolution , in cont a dimer . The SEC species . This siSteccherinum murashkinskyi laccase recorded with different degrees of X-ray exposure to obtain detailed information on the structural variations in the TNC site and the reaction mechanism [MtL structure contains three fully occupied copper ions in the TNC-site. However, even the dataset that displayed the least radiation damage, revealed some disorder in this site. The site was modelled as a classical resting state with oxidized Cu ions, a hydroxide ion bridging the T3-coppers and a water/hydroxide ion coordinated to the T2-Cu , His98 was found in its oxidized form, as an oxo-histidine, and in a conformation that blocked the T2-channel [MtL, where the electron density is also consistent with the presence of an oxo-His98, but in MtL the residue is only partially oxidized and/or adopts multiple conformations.The TNC-site is connected to the exterior through the T2-solvent channel . In the -channel . His98 iin vivo or in biotechnological processes, not only the catalytic machinery involving the Cu-centres, but also the surface of the protein plays a role in enzyme function. Obviously, surface residues surrounding the T1-substrate binding pocket could affect substrate specificity, but also more distant sites may play important roles. Assuming that conserved residues play a role in biological function, we have investigated the sequence conservation among asco-laccases. The overall sequence conservation between the five asco-laccases with known structures is only 23%  close to the entrance to the T1-pocket that confers discrimination against phenolic acids as substrates due to electrostatic repulsion [AnL is in many ways a deviating asco-laccase and an example of how substrate selectivity may be achieved through evolution of surface residues surrounding the T1-pocket. An observation that could be exploited in rational enzyme engineering.For proteins, appearances matter. Thus, for laccases that act on large, complex lignocellulose substrates either  regions . The fouion Figs  and 7E. epulsion . AnL is Cerrena maxima [MtL contains 11 tyrosine residues, four of these are located on the surface and either conserved or replaced by Phe in the other four asco-laccases, except for Tyr416 that is only conserved in the MtL-MaL-TaL subdivision . At some sites, only the innermost N-acetylglucosamine (NAG) moiety could be modelled, whereas oligosaccharides containing five and six sugar rings, respectively, were visible at Asn61 and Asn201. The glycan linked to Asn61 is stabilized by crystal packing and only the innermost NAG-residue is involved in polar interactions with the protein. In contrast, all six sugar rings of the glycan attached to Asn201 interact with the protein via hydrogen bonds, hydrophobic interactions and/or water mediated contacts. This oligosaccharide packs against the protein in a long groove on the surface, at the interface between domain A and B . However, in this case the glycan displays only few interactions with the protein, and its role is unclear. The sites at positions Asn216, Asn289 and Asn396 are additionally conserved within the MtL-MaL-TaL subdivision. In particular, the two NAG-NAG residues linked to Asn289 are highly integrated in the structure, they form almost identical interactions in all three laccases, and are thus likely to be involved in the stabilization of two loops in domain B. The glycan at Asn216 stabilizes a glycine-rich loop in MtL and MaL. In TaL, the composition of the loop is different  in the crystal structure of deglycosylated BaL , the noted differences in the architecture of the T1-Cu pocket suggests some variability in the preferred substrates for the individual laccases. Our comparative study of asco-laccases shows an abundance of conserved N-glycosylation sites, which are spatially close and located in a surface area that is also rich in other conserved residues. This gives unique characteristics to this part of the surface which is likely to influence interactions with large biopolymers and may play a role in interactions with carbohydrate-rich substrates like lignocellulose. To understand laccase function in detail, it is therefore important that surface properties like glycosylation and electrostatics are taken into consideration.S1 FigMtL monitored as decay in activity after incubation at 65°C. Laccase activity was measured in 50 mM acetate buffer pH 4.5 using ABTS as substrate. The change in absorbance over time was measured at 415 nm to follow the formation of a blue-green product, thought to be the radical ABTS+ [1/2) around 140 min.The thermal stability of al ABTS+ . For staal ABTS+ . The abs(TIF)Click here for additional data file.S2 FigThe large single crystal was one of the crystals used for data collection (approximate dimensions 700 x 200 x 30 μm).(TIF)Click here for additional data file.S3 FigClassical resting state geometry with oxidized Cu ions, OH- bridging the T3-coppers and water/hydroxide coordinated to T2. The Cu-Cu-distance is 4.6 Å.(TIF)Click here for additional data file.S4 FigMapping of N-glycosylation sites in selected asco- (A) and basi- (B) laccases. Structure-based alignment where putative N-glycosylation sites with the consensus motif Asn-X-Ser/Thr are highlighted with orange boxes. For sites where attached glycans are observed in the crystal structure, the box is filled. Sites that are unlikely to be glycosylated, as they are either buried inside the protein structure or contain a Pro residue at the X1 site (Asn-Pro-Thr/Ser), are indicated with boxes with a dotted line. The alignment was made with Promals3D [Pycnoporus cinnabarinus , Rigidoporus microporus , Trametes sanguinea , Lentinus sp. , Trametes versicolor , Lentinus tigrinus , Trametes hirsuta , Cerrena maxima , Trametes versicolor , Trametes trogii , Coriolopsis gallica , Trametes ochracea , Coprinus cinereus  and asco-laccases from Myceliophthora thermophila , Melanocarpus albomyces , Thielavia arenaria , Botrytis aclada  and Aspergillus niger . No glycans were modelled in the RmL structure (1V10), but two sites were identified by MS-analysis [TsL (UNIPROT C1JCL7), but this sequence is included as this is the laccase used in the (de)-glycosylation studies by Vite-Vallejo and coworkers [romals3D , 43 and analysis . No crysoworkers .(TIF)Click here for additional data file.S5 FigN-glycosylation sites A) Asn61, B) Asn88, C) Asn201, D) Asn216, E) Asn289, F) Asn376 and G) Asn396.(TIF)Click here for additional data file.S1 Table(PDF)Click here for additional data file.S2 Table(PDF)Click here for additional data file.S3 Table(PDF)Click here for additional data file.


To study the clinical significance of the Graves' orbitopathy-specific quality of life (GO-QOL) questionnaire in mainland Chinese patients.  A cross-sectional study was performed at the Ophthalmology Department of the Sun Yat-sen Memorial Hospital from April 2017 to April 2018. Eighty-eight consecutive Graves' orbitopathy (GO) patients completed the two subscales of the GO-QOL questionnaire: visual functioning and appearance. The disease severity of GO was measured by the European Group on Graves' Orbitopathy (EUGOGO) classification, and clinical activity was evaluated by the clinical activity score (CAS). p < 0.05). Lower QOL scores for appearance were significantly correlated with the CAS (p < 0.05). Although no correlation was found between the appearance subscale scores and disease severity (p=0.407), a downward trend in the appearance subscale scores as the severity of GO increased from mild to sight-threatening GO was found.  The mean scores of GO-QOL questionnaire for the visual functioning and appearance subscales were 68.4 ± 31.2 and 62.0 ± 27.4, respectively. Lower QOL scores for the visual functioning subscale were significantly correlated with disease severity, the CAS and diplopia (all  A strong correlation between disease severity and clinical activity has been shown in the GO-QOL questionnaire, suggested by the EUGOGO. The GO-QOL questionnaire is a simple and effective appraisal instrument in the evaluation of health-related QOL in the mainland Chinese patients with GO. Graves' orbitopathy (GO) is an autoimmune inflammatory disease that potentially causes sight-loss, disfigures appearance, and results in an obvious loss of quality of life. –3 About The Graves' orbitopathy-specific quality of life (GO-QOL) questionnaire was specifically created for GO patients by Terwee et al. and his colleagues.  The quesThis cross-sectional study included 88 patients with GO who were followed up at the Sun Yat-sen Memorial Hospital, Sun Yat-sen University, Guangzhou, China, from April 2017 to April 2018. GO was diagnosed in the Department of Endocrinology and Ophthalmology based on the presence of typical eye signs and symptoms in a patient with Graves' disease. All clinical observations and objective measurements were consistently performed by one ophthalmologist (Mei Wang). Data collected included patients' age, sex, signs, symptoms, duration of GO, clinical activity, and severity classification of GO according to the European Group on Graves' Orbitopathy (EUGOGO), history and duration of thyroid disease, history of smoking, and other systemic and eye diseases. Ophthalmological data included a measurement of best-corrected visual acuity, a slit lamp examination of the anterior eye segment, and a fundus examination with a 90D lens. Proptosis was measured with a Hertel exophthalmometer. This study was conducted in accordance with the tenets of the Declaration of Helsinki, the protocol of this study was approved by the Sun Yat-sen Memorial Hospital, Sun Yat-sen University Committee, and informed consent was obtained from all participants.The English version of the GO-QOL questionnaire was downloaded from the EUGOGO website and then translated into Chinese.  The quesThe inflammatory activity of GO, including (1) spontaneous retrobulbar pain, (2) pain on attempted upward or downward gaze, (3) redness of the eyelids, (4) redness of the conjunctiva, (5) swelling of the caruncle or plica, (6) swelling of the eyelids, and (7) swelling of the conjunctiva (chemosis), was measured by the clinical activity score (CAS).  The CAS EUGOGO classification  was usedp-value less than 0.05 was considered statistically significant.Data were collected and entered into a computerized statistical software package (SPSS 22.0) in a standard fashion. Continuous variables are expressed as the means ± the standard deviations, and categorical variables are presented as percentages. The floor and ceiling effects of the GO-QOL were measured in terms of the percentage of GO patients. In previous studies, “significant” floor and ceiling effects were defined as >15% of patients and “substantial” floor and ceiling effects were defined as >30% of patients. , 17 ThesDemographic and clinical data for all patients are shown in The percentages of responses to each item of the GO-QOL are summarized in p=0.003) and disease severity (p=0.002) . The visual functioning subscale scores were markedly higher in patients with mild GO (84.1 ± 18.2) compared to those with moderate-severe (65.2 ± 31.1) and/or sight-threatening GO (51.6 ± 35.3). The scores of GO-QOL visual functioning subscale were significantly different between mild versus moderate-severe GO (p=0.006) and mild versus sight-threatening GO (p=0.001). There were no differences between patients with moderate-to-severe versus sight-threatening GO (p=0.225). Additionally, a significant correlation was found between visual functioning scores and sex and diplopia (p < 0.001). However, there was no difference between visual functioning scores and age (p=0.214) or proptosis (p=0.546) in this study , watching TV (58.0%), doing something you wanted to do (50.0%), and hobbies or pastimes (46.6%). In this study, the visual functioning subscale scores of GO-QOL were markedly related to clinical activity  (p=0.407)(p=0.123), age (p=0.800), diplopia (p=0.696), or proptosis (p=0.261) in this study , “stared at in the streets” (62.5%), and “influence on self-confidence” (59.1%). The appearance subscale scores were markedly higher in patients with mild GO (66.6 ± 24.8) compared to patients with moderate-severe (62.4 ± 26.2) and/or sight-threatening GO (54.5 ± 31.0). The appearance subscale scores of GO-QOL were closely related to clinical activity (p=0.001) . This st(p=0.407). Additiois study .GO restricted patients' daily activities such as reading, watching television, and enjoying free time, as well as created a dysfunction in social roles and an impaired self-confidence because of the altered appearance. The GO-QOL survey was devised by Terwee et al.  and has The mean GO-QOL scores measured in this study were much higher than those measured in the Taiwanese (Chinese) survey . The difference in QOL scores might be associated with different patient compositions.Female patients attained higher visual functioning and lower appearance scores in this study compared with male patients. We speculate that physical attractiveness might be more important to women than it is to men among Chinese people. However, visual function might be more important to men than it is to women. Moreover, no relation was found between appearance score and age or visual functioning score and age. However, the results implied that patients aged 13–24 years attained lower appearance scores than older patients. One possible reason was that younger patients are vulnerable to the negative effects of GO on their emotions and appearance, which lead to a dysfunction in social roles and an impaired self-confidence.Previous studies reported that significant ceiling effects were found on the two subscales and concluded that the GO-QOL lacked the sensitivity to distinguish an effect in mild GO patients. As we have known, a significant ceiling effect would limit the ability to measure improvements in the GO-QOL in GO patients. In this study, we found that no significant ceiling and floor effects were found on the two subscales of the GO-QOL. This result was similar to the Taiwanese study.  Many stuOur research has some limitations. First, this was a cross-sectional study, which makes it hard to assess dynamic changes in a patient's GO-QOL score over time. Second, GO severity and activity were evaluated by one ophthalmologist, which could improve reliability but might create systematic bias. Third, all participants were recruited from a single academic institution, and the backgrounds of participants in our clinic might be different from those of patients in a community setting.In conclusion, GO could interfere with patient quality of life by damaging visual function and harming appearance. The GO-QOL survey is a simple and valuable tool to evaluate the severity and activity of GO in the mainland Chinese patients.


In order to understand how NK cells combine different cell death mechanisms, it is important to quantify target cell responses on a single cell level. However, currently existing reporters do not allow the measurement of several protease activities inside the same cell. Here, we present a strategy for the comparison of two different proteases at a time inside individual target cells upon engagement by NK cells. We developed single-fluorescent protein reporters containing the RIEAD or the VGPD cleavage site for the measurement of granzyme B activity. We show that these two granzyme B reporters can be applied in combination with caspase-8 or caspase-3 reporters. While we did not find that caspase-8 was activated by granzyme B, our method revealed that caspase-3 activity follows granzyme B activity with a delay of about 6 min. Finally, we illustrate the comparison of several different reporters for granzyme A, M, K, and H. The approach presented here is a valuable means for the investigation of the temporal evolution of cell death mediated by cytotoxic lymphocytes.Natural killer (NK) cells eliminate infected and tumorigenic cells through delivery of granzymes  In the second mechanism, CD95L or TRAIL are presented at the surface of NK cells and induce extrinsic apoptosis in target cells through activation of the death receptors CD95 or TRAIL-R1/-R2  cells can eliminate infected and tumorigenic cells . To do sL-R1/-R2 , 5.How NK cells orchestrate the activities of granzymes and the activation of extrinsic apoptosis remains poorly understood. Extrinsic apoptosis starts with the formation of the so-called death-inducing signaling complex, composed of activated death receptors and recruited FADD adaptor proteins and initiator procaspases-8/-10. Once activated, these caspases cleave and activate effector procaspase-3/-7 , 7, leadin vitro have been reported to be more efficiently cleaved, for example, DNA-PKc or BID  different cleavage sites for different proteases or of (ii) different cleavage sites for the same protease.In order to establish single-fluorescent protein reporters for the measurement of granzyme B activity, we designed and tested two different reporters carrying a linker sequence known to be cleaved by granzyme B. The first reporter carries the amino acid sequence RIEADS (single amino acid code), which is present in the protein BID. The second reporter carries the amino acid sequence VGPD from the protein DNA-PKc as cleavable linker. We co-expressed the two reporters NES-RIEADS-mCherry and NES-VGPD-mGFP in HeLa cells expressing CD48, a ligand for the activating NK cell receptor 2B4 CD244), which renders them more sensitive to killing by NK cells . The tra, which rCD95 signaling in HeLa cells leads to notable caspase-8 and -3 activation . On the Therefore, we tested if caspase-8 and granzyme B activity can be distinguished within the same cell using our single-fluorescent protein reporters. For this aim, we measured NES-ELQTD-mGFP (for caspase-8), together with either NES-RIEADS-mCherry or NES-VGPD-mCherry (for granzyme B). Upon addition of soluble trimerized CD95L (IZsCD95L) to HeLa cells, we observed efficient cleavage of the caspase-8 reporter NES-ELQTD-mGFP. In contrast, in the same cells, the NES-RIEADS-mCherry reporter was cleaved only up to 10% Figure A and thevia cytotoxic granules) and caspase-8-dependent (via CD95) pathways to kill target cells.Transient expression of shRNA against CD95 led to a reduction of CD95 protein expression on the surface of HeLa cells Figure A. The nuIt is known that effector caspase-3 activation can be a consequence of granzyme B activity . We thusNatural killer cells notably express granzyme A and B , 46, butThe single-fluorescent protein reporters employed in this study are part of a larger panel of protease reporters, based, for example, on cyclic luciferase , 48, fluWe demonstrate our approach using HeLa cells as targets as they are known to be responsive to death receptor ligands , 51 and We showed that the reporters developed here containing the RIEADS or the VGPD cleavage site are suitable for the measurement of granzyme B activity. We furthermore systematically tested putative reporters containing amino acid sequences that were previously shown to be cleaved by granzyme A, B, M, H, and K . PrimaryOur experiments have demonstrated that granzyme B reporters can be applied in combination with caspase-8 or caspase-3 activity reporters. Strong caspase-3 activity correlated with granzyme B activity. These results support earlier studies , 27, 28 Human CD48 (UniProtKB P09326), human serpin B9/PI-9 (from Gateway cDNA library of the DKFZ), and IZ-sCD95L  were subl-glutamine, 50 µM 2-mercaptoethanol, 12.5% horse serum, 12.5% fetal bovine serum, and penicillin/streptomycin, 100 µg/ml each (Invitrogen). Human NK cells were isolated from PBMCs using the Dynabeads Untouched Human NK Cell kit (Thermo Fisher Scientific) according to the manufacturer’s instructions. NK cells were between 90 and 99% CD3−, CD56+, and NKp46+ as assessed by flow cytometry. NK cells were expanded in 96-well round-bottom plates (Nunc) with irradiated K562-mbIL15-41BBL feeder cells (kind gift from Dario Campana) in IMDM Glutamax supplemented with 10% FCS and 1% penicillin, IL-2 , and IL-15  at 37°C in a humidified 5% CO2 incubator. IL-21  was added on the first day. Soluble CD95 ligand fused to the isoleucine-zipper domain  was produced in 293T cells, which were seeded in 6-well plates. 24 h after cell transfection using JetPrime reagent (Polyplus), supernatant was replaced by fresh medium. At the next day, supernatant containing secreted ligand was harvested.HeLa and MDA-MB-468 cell lines were maintained in Dulbecco’s modified eagle medium  containing 10% fetal calf serum , penicillin/streptomycin, 100 µg/ml each (Invitrogen) and 1% non-essential amino acids plus 1 mM Sodium Pyruvate (Gibco) for MDA-MB-468 only. HeLa (CD48) cells over-express CD48 and were maintained in medium supplemented with 0.5 µg/ml puromycin (Sigma-Aldrich). NK92-C1 that stably express IL-2 were maintained in phenol red-free Minimum Essential Medium Eagle Alpha  without ribonucleosides and deoxyribonucleosides but containing sodium bicarbonate, supplemented with 2 mM 4 to 1.8 × 105 NK cells per well. One field of view in confocal microscopy contained about 2 to 6 transfected cells. All cells expressing both reporters were analyzed. The time-resolution was about 2–4 min depending on the number of imaged fields and microscopy settings. Equal target cell preparation, equal NK cell preparation , and parallel measurement of different wells of an 8-well chamber allowed comparison of different conditions in one experiment. Time-series data were acquired for up to 12 h.The presented approach uses fluorescence microscopy, which allows the extraction and correlation of several features, including the time of cell death. Time-lapse microscopy was performed with the TCS SP5 confocal laser scanning microscope from Leica  equipped with a 63×/1.4 OIL, HCX PL APO CS objective. We used the live data mode of the Leica software for autofocusing as described in Ref. . With thhttps://github.com/jbeaudouin/NK_cell. Alternatively, we suggest using the background subtraction function in the “Process” tab provided in ImageJ or Fiji, which is based on the rolling ball algorithm. In most cases, two reporters were measured within one cell: to analyze them, one channel was assigned green, the other red, both were transformed into RGB, and they were then superimposed. The mean intensity within the nucleus was quantified by choosing a representative region within the nucleus at each time point until cell death was observed. We chose to normalize the nuclear intensity to the cytosolic intensity at each time point for soluble-cytosolic reporters to calculate the percentage of reporter cleavage.Images were analyzed using ImageJ . To quanOpen image stack of the first channel (time-series of one field of view) in ImageJ or Fiji. If applicable, open image stack of the second channel (time-series of one field of view) in ImageJ or Fiji. At this stage, one works possibly with 8-bit or 16-bit—type images, depending on the microscope settings.Subtract the background.For two reporters measured: assign the stack with the channel of the first reporter in red (lookup table red), and the stack with the channel of the second reporter in green . At this stage, you still work with 8-bit or 16-bit—type images.For two reporters measured: set the scale of the two stacks equal, meaning that the values in the 8-bit images would be set from 0 to 255, or values in the 16-bit images would be set from 0 to 65535.Transform the stacks into RGB-type. Note that after this transformation, the intensity range is from 0 to 255.Create an RGB-stack by adding the two stacks. For this use, the “Image Calculator” function in the “Process” tab of ImageJ or Fiji.Save this stack.Choose a region of interest using the rectangle or oval selection tool. Select a representative region within the nucleus or the cytosol, respectively.https://github.com/jbeaudouin/NK_cell). In the results table, a row containing three values for the mean intensity signal in the red channel, green channel, and the blue channel of the selected region of interest will be displayed. To call this plugin, please create a shortcut on the keyboard (Plugins tab > Shortcuts > Add shortcut)Measure the mean signal of this selection using the plugin RGB_Measure3  for further data analysis. Annotate the date of the experiment and experimental conditions.The signal of the nucleus is then normalized (divided by), the signal measured in the cytosol, as explained in the first section of the results.Despite the background subtraction, it may be useful to subtract a remaining offset. This signal may originate from signal in the nucleus at time 0 (before addition of NK cells).The detailed image analysis workflow is as follows:Cell death of HeLa target cells could be determined by the change of the cell’s morphology. While cell rounding was often observed, we found also cells showing shrinkage without typical apoptotic blebbing. Changes in target cell morphology were unambiguosly detected by monitoring living target cells by time-lapse microscopy in the fluorescence and transmission light channels.P values were greater than 0.05 (ANOVA). Boxplots additionally show raw data points. Boxes indicate median, 25% quartile, and 75% quartile. Whiskers show minimal and maximal values. To plot the mean kinetics of reporter cleavage of single cell data, we set the time point that directly preceded cell death to time = 0 and calculated the mean and SD of single cell responses. The time values can, therefore, have a negative sign on the plots.Data were visualized and analyzed using OriginLab software. Data groups were considered significantly different as indicated in the text, but considered not significantly different when CL designed the study, performed experiments, analyzed the data, and wrote the paper. PS, IP, and DU performed experiments and analyzed data. MC isolated and purified primary human NK cells. RE, JB, and CW supervised the work and helped writing the paper.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


No difference was observed between the pre-ictal and ictal periods. Control subjects demonstrated intermediate Brain Engagement Index values, that is, higher than inter-ictal, yet lower than pre-ictal. Our preliminary results demonstrate the potential advantage of the use of a simple EEG system for improved prediction of migraine attacks. Further study is required to evaluate the efficacy of the Brain Engagement Index in monitoring the migraine cycle and the possible effects of interventions.Migraine attacks can cause significant discomfort and reduced functioning for days at a time, including the pre-ictal and post-ictal periods. During the inter-ictsal period, however, migraineurs seem to function normally. It is puzzling, therefore, that event-related potentials of migraine patients often differ in the asymptomatic and inter-ictal period. Part of the electrophysiological dynamics demonstrated in the migraine cycle are attention related. In this pilot study we evaluated an easy-to-use new marker, the Brain Engagement Index (BEI), for attention monitoring during the migraine cycle. We sampled 12 migraine patients for 20 days within one calendar month. Each session consisted of subjects’ reports of stress level and migraine-related symptoms, and a 5 min EEG recording, with a 2-electrode EEG device, during an auditory oddball task. The first minute of the EEG sample was analyzed. Repetitive samples were also obtained from 10 healthy controls. The brain engagement index increased significantly during the pre-ictal ( Migraine is a prevalent disease, affecting more than 15% of females and more than 5% of males . During The mechanism responsible for reduced habituation during the inter-ictal phase is not clear. It has been suggested that it is an epiphenomenon of reduced response in the initial sample blocks, which sets limits upon the possible range of habituation ,13,14. TOver the last several years we have developed an effective single-electrode marker for attention. Our work shows that monitoring attention processes can be efficiently done over a wide frontocentral area, including prefrontal electrodes ,16. In lFurthermore, we simplified the EEG analysis to adjust the extraction of relevant attention-related markers from a short sample, at the scale of 1 min, on the basis of template matching ,19 of thBased on this method, in this work we evaluated the applicability of following the dynamics of this simple marker during the migraine cycle. We hypothesize that, based on template matching, analyzing the attention-based markers from 1 min samples will distinguish between inter- and pre-ictal (12–48 h prior to the attack) electrophysiological patterns of cortical activity in migraine patients. The use of only the first minute of the sample aligns with the hypothesis cited above, in that the ERP differentiation and dynamics in migraine stems from changes in the response in the initial sample blocks. Such easy-to-extract attention-based markers have potential applications in the prediction of migraine attacks. Nevertheless, this is merely a pilot study and its results can only be viewed as preliminary.Thirteen migraine patients were recruited to participate in this study. Of these, 12 completed all 20 sessions and as such comprise the patient group; 1 patient left mid-study due to personal reasons and their data was excluded from the analysis. Patients were recruited through word of mouth and snowball sampling, starting with individuals who had contacted the headache clinic at Rambam Health Care Campus. The inclusion criteria were suffering from 2 to 10 attacks per month, and the commitment to participate in at least 20 sampling sessions within one calendar month. The exclusion criteria included any neurological or psychiatric disorders, presence of other chronic pain or hearing disorder and/or known eardrum injury. All patients underwent neurological evaluation by a migraine specialist for diagnosis verification. The patients maintained their regular preventive (if any) and acute treatment regimens during the sampling period. The study on migraine patients was performed in the Laboratory of Clinical Neurophysiology, Technion Faculty of Medicine and Rambam Health Care Campus. The data from migraine patients were compared with the samples of ten control subjects. The exclusion criteria of the control subjects included diagnosis of psychiatric or any neurological disorder, recent history of drug or alcohol abuse or dependence and hearing disorder and/or known ear drum impairment. These subjects were sampled twice a week for 8 weeks. Seven of the control subjects were tested in the Emotion–Cognition Research Center, Shalvata Mental Health Care Center, Hod-Hasharon, affiliated with the Sackler Faculty of Medicine, Tel-Aviv University. Three other subjects were tested in their homes after being evaluated by a physician and signing informed consent at either Shalvata Mental Health Center or Emek Medical Center.The final study group consisted of 12 migraine patients , 20–35 years; 11:1 F:M. The mean disease duration was 9.8 ± 6.3 years. The mean and maximal pain during a migraine attack were 6.0 ± 1.3 and 8 ± 1.3, respectively (VAS 0–10). The monthly attack frequency ranged from 2 to 10 (mean 4.7 ± 2.1). The number of migraine days per month varied from 2 to 15 days (mean 6.4 ± 3.5). Seven out of 12 patients had migraine with aura. Two patients had visual aura, 5 patients experienced signs of both visual and sensorimotor aura. During an attack, all patients had phonophobia, 11/12 had photophobia, 6/12 suffered from increased olfactory sensitivity, 2/12 suffered from increased taste sensitivity, 5/12 experienced vomiting and/or nausea. None of the patients utilized daily preventative treatment. Due to the small sample size we did not differentiate in the analysis between migraines with and without aura.The study on migraine patients was approved by the local Helsinki Committee of Rambam Health Care Campus, approval number 0358-15-RMB. The study on control subjects was approved by the local Helsinki Committee of Shalvata mental health center, approval number 0010-15-SHA. All patients provided signed inform consent prior to participation in the study.EEG data were recorded from the NeuroSky MindWave single-channel system , with one frontal electrode (~Fpz) and one reference electrode on the earlobe, at a sampling rate of 512 Hz. The MindWave EEG headset uses dry EEG electrodes. Sampled data were transferred through a wireless connection to the experiment computer for offline processing. Each sampling session involved 5 min of stimulus-free recording and 5 min of recording that was synchronized with an auditory oddball protocol. The oddball stimuli consisted of 1000 and 2000 Hz tones of 40 ms duration, presented binaurally at ~60 dB. The stimuli comprised a frequent tone (1000 Hz) presented 80% of the time, and a rare tone (2000 Hz) presented 20% of the time. The inter-stimulus interval was selected randomly within the range of 2 to 3 s. Before each sampling period, good skin contact was secured (the headset provides an index of contact level). Only data from the 1st minute of the stimulus-related samples were used in the analysis. The use of data from only the first minute was done in continuation of previous studies with other clinical populations ,19. It sAll patients underwent 3–5 sampling sessions per week, with 24–72 h between subsequent sessions. At the beginning of each sampling session, the patient completed a questionnaire which addressed the following parameters: (a) Indication of whether a new attack took place since the previous sampling session, and if yes, an indication of the timing of the attack; (b) indication of whether pre-ictal (prodrome) and post-ictal (postdrome) symptoms were noted since the previous sampling session ; (c) assessment of stress level on a visual analog scale (VAS), ranging from 0 (no stress) to 10 (severe stress). The averaged ERP template  is emphaWe computed the Brain Engagement Index for data from the 1st minute of the stimulus-related sample. The computation is based on template matching , a technBased on large single-trial variability , matchinThe sampling days of each patient were categorized retrospectively, according to the reported times of attacks, as follows: (a) Ictal samples during which the patient reported an ongoing attack; (b) pre-ictal samples that preceded an attack by up to 48 h ; (c) post-ictal samples that followed each attack by up to 48 h ; (d) inter-ictal samples, which do not match the ictal, pre-ictal, or post-ictal categories described above. If a sample was overlapping , it was excluded from analysis. Control subjects were sampled 14–16 times over a period of 8 weeks (1–2 times each week).t-test by subject for comparing the average BEI in the 4 periods . We further compared the 4 periods with the samples of the control subjects. Statistical significance was defined as p ≤ 0.05. In addition, we evaluated the changes of the post-ictal EEG dynamics by comparing the 1st and 2nd post-ictal days with a one-tailed, paired sample t-test.The focus of analysis was on the changes between the inter-ictal and the pre-ictal period. We also compared the ictal period and the inter-ictal and pre-ictal periods, as well as the post-ictal period to all other periods. Statistical significance for sample-based analysis was analyzed using repeated measures ANOVA corrected for multiple comparisons. For subject-based analysis we used paired t-test. For the stress VAS evaluation, we used the absolute difference between the stress VAS report on each sampling day and the report on the previous day. We focused on absolute difference in stress VAS because it yielded some differentiation between the inter-ictal and the pre-ictal periods. Such differentiation was not evident by averaging the raw VAS scores or by averaging the non-absolute VAS differences. Therefore, we averaged the absolute VAS difference for the inter-ictal period and pre-ictal period for each subject. We then compared the inter-ictal and pre-ictal averaged differences across subjects and evaluated the significance of this comparison using t-test.Behavioral data from the inter-ictal and the pre-ictal periods were compared based on the onset of new pre-ictal symptoms and on measuring differences in VAS reports of stress. The onset of new pre-ictal symptoms on a specified sampling day was defined as such if symptoms were newly reported, and had not been reported during the previous sampling day. We focused on newly reported symptoms because this yielded some differentiation between the inter-ictal and pre-ictal periods. Such differentiation was not evident by simply comparing the ratios of days with reported symptoms, without consideration of whether the symptoms were also reported in the preceding days. To that aim, we calculated the ratio between the days with newly reported symptoms and the total number of sampling days (“report ratio”) in the inter-ictal period and in the pre-ictal period for each subject. We then compared the inter-ictal and pre-ictal report ratios across subjects and evaluated the significance of this comparison using a After noisy samples were automatically excluded, the analysis was based on 43 inter-ictal (3.58 ± 1.38 per subject), 19 pre-ictal (1.58 ± 1.24 per subject), 13 ictal (1.08 ± 1.24 per subject), and 22 post-ictal samples . For the control subjects, 99 non-noisy samples were included. The control subjects were comprised of two age groups: 5 subjects aged 23–32 (3:2 F:M), which were matching in age with the migraine subjects; and 5 subjects aged 59–75 (3:2 F:M).p = 0.34). Similarly, no differences in stress VAS between pre- and post-ictal phases were found (p = 0.16). No significant difference was observed for the onset of newly detected symptoms between pre- and post-ictal migraine phase  and ictal  periods. The difference between the pre-ictal and ictal periods is not significant. The two post-ictal days did not differ significantly from the two pre-ictal samples (p = 0.09), ictal samples (p = 0.16) and inter-ictal samples (p = 0.08). Control samples differed significantly from the pre-ictal samples (p < 0.01), but not from the inter-ictal (p = 0.06), ictal (p = 0.09) and post-ictal samples (p = 0.51). In Lower BEI values were observed in the inter-ictal compared with the pre-ictal (p = 0.35). Therefore, we used data for the entire control group to compare with the migraine samples.p < 0.001). Post-hoc Tukey analysis indicated a significant difference in Brain Engagement Index values between the inter-ictal vs. pre-ictal periods (p = 0.001). The difference between the inter-ictal and ictal periods was also significant with Tukey HSD correction (p = 0.020). There was no significant difference between the pre-ictal and ictal periods (p = 0.27). The post-ictal period did not differ significantly from the pre-ictal (p = 0.09), inter-ictal (p = 0.08) and ictal (p = 0.16) periods. The control samples differed significantly from the pre-ictal samples (p < 0.01), but not from the inter-ictal (p = 0.06), ictal (p = 0.09) and post-ictal samples (p = 0.51).The main focus of the study considered the possibility of identifying changes between the inter-ictal and pre-ictal and ictal periods. The difference between these periods was significant according to repeated measures ANOVA  = 8.57, t-test analysis of paired subject averages was significant between the inter-ictal and pre-ictal periods (p = 0.008), and between the inter-ictal and ictal periods (p = 0.003). There was no significant difference between the pre-ictal and ictal averages (p = 0.26). No significant differences were found between the paired post-ictal and inter-ictal samples, post-ictal and ictal samples, or pre-ictal and ictal samples . The bottom inset of In line with ANOVA results, the subjects-based paired t-test .The baseline likelihood of an attack in 48 h is ~25%, taking into account the frequency of attacks averaged over all patients. The result of a single BEI test improves attack prediction, and the results of two tests, taken one day after another, improves prediction further. A BEI value above threshold is denoted by “+”, and “–” denotes BEI values below threshold. The threshold is 0.5 for 10 out of the 12 patients. In 2 patients the average inter-ictal BEI was ~0.75 . For these patients, a threshold of 0.75 was selected. Only tests from the inter-ictal and pre-ictal periods were included in the prediction analysis. Missing tests are due to noise or skipped sampling days. In the double (2 day) test shown in We calculated the predicted accuracy for the pre-ictal and ictal periods. Consistent with previously reported work ,9,10,11,We suggested in earlier studies that the Brain Engagement Index is related to attention and increases with increased attention ,16,18,19Reduced habituation of early ERP waves was demonstrated in migraine for multiple sensory modalities ,5. Some Previous works have reported pseudo-normalization of ERP-based electrophysiological markers in the transition from the inter-ictal to the pre-ictal period ,11. We oIn a detailed theoretical work , we suggOur behavioral results demonstrated only a modest and non-significant change in self-reported stress in the transition from the inter-ictal to the pre-ictal period. This, however, may be the result of limited sensitivity when measuring stress on a VAS scale. We did not observe a clear tendency for increased stress in the inter-ictal—pre-ictal transition, and a decrease in stress was reported as frequently as an increase. Others have observed an occasional decrease in stress in the inter-ictal—pre-ictal transition. This phenomenon, termed “stress letdown” , is consIrrespective of the correctness of this or any other theory, the present work demonstrates the preliminary feasibility of migraine attack prediction with an easy-to-use electrophysiological tool. Such a tool may also make it possible to evaluate preventive interventions, which often consist of non-medical stress-reducing techniques. Nevertheless, it should be stressed that the current study is based on small groups of patients and controls and can only be viewed as a pilot which provides further supporting evidence regarding the potential feasibility of an easy-to-use tool for attention measuring, which is additive to previously published studies ,19 with


Introduction: Ageing is associated with several physical, psychological, and behavioral changes. These changes are closely related with general health problems and quality of life in old age. The CASP-19 multidimensional instrument was specially designed to measure quality of life in the elderly. The different language versions of this scale have been used in more than 20 countries. However, Lithuanian translation was not available. The objective of our study was to test psychometric properties of the Lithuanian version of the CASP-19 questionnaire. Materials and methods: A cross-sectional study was performed with ambulatory men and women aged 60 and older, living in a community in Vilnius, Lithuania. Exclusion criteria were current acute illness, malignant tumor, and Mini-Mental State Examination (MMSE) score < 25 points. Psychometric properties of CASP-19 were tested using reliability and validity methods. Results: The study sample consisted of 132 participants, 28 (21.8%) of them were men and 103 (78.2%) women. Analysis of psychometric properties of the Lithuanian version of CASP-19 showed high internal consistency , good agreement between test-retest measures with an ICC of 0.82 (95% CI 0.79–0.85) and good convergent and divergent construct validity. Conclusions: The psychometric properties indicated that the Lithuanian version of CASP-19 was reliable and valid. As such, it might be used to evaluate quality of life in elderly people. One of the negative consequences of the ageing of the global population is the increase in health problems and illnesses, which may soon overwhelm health systems in all countries. Quality of life (QoL) in later life has become a research target and global policy issue . QoL is The CASP-19 multidimensional instrument was designed to measure a quality of life in the elderly. The 19 item questionnaire is composed of four domains, and the first initial of each domain makes up the acronym—Control, Autonomy, Self-realization, and Pleasure . Doyal aThe CASP-19 has been used in more than 20 national and international studies. It has already been translated into 16 languages. This tool has been included in the Survey of Health, Aging, and Retirement in Europe (SHARE) , the EngThe aim of our study was to test psychometric properties of the Lithuanian version of the CASP-19 questionnaire.The CASP-19 questionnaire consisted of 19 items. For each of these items, participants were asked to rate how often a statement described how they felt, for example, “My age prevents me from doing the things I would like to do,” or, “I enjoy the things I do.” Using a 4-point Likert scale, a participant would respond with one of the following options: often = 0, sometimes = 1, rarely = 2, or never = 3. Thirteen positively worded items  were reverse-coded. The total CASP-19 score ranged from 0 to 57; higher scores indicated higher QoL. The CASP-19 was a self-administered questionnaire, and could be completed in approximately 10–15 min [The Lithuanian translation of CASP-19 was performed according to guidelines for the process of cross-cultural adaptation of self-report measures  and the Stage 1:Initial translation. An initial translation from English to Lithuanian was performed by two independent bilingual translators, both of whom were native Lithuanian speakers. Translator 1 had a medical background and knowledge about health care terminology. Translator 2 had no knowledge about medical terminology or the construct of the instrument.Stage 2:Synthesis of the translations. A consolidation of these two translations in which a consensus was reached and a preliminary initial translated version of the instrument in the target language was generated.Stage 3:Backward translations. The preliminary initial translated version was backward translated into the original language by two more independent bilingual translators, both native English speakers with no medical background or knowledge about the original English version of the CASP-19 questionnaire.Stage 4:Expert committee review. The expert committee included one methodologist (the investigator of a research team who is familiar with content areas of the construct of the instrument), one linguistic specialist, and four translators who had performed translation and back-translation. The expert committee compared two back-translations with the original questionnaire and reviewed the grammatical structure of the sentences and similarity in meanings. Expert committee members whose mother language was Lithuanian enhanced the quality of the pre-final version of the translated instrument. Any ambiguities and discrepancies regarding cultural meaning and colloquialisms or idioms in words and sentences of the questionnaire were discussed and resolved through consensus of a pre-final version of the questionnaire.Stage 5:Test of pre-final version. Pre-testing of the CASP-19 pre-final version was performed to ensure good comprehension of each question. Fifteen individuals were asked to rate items using a dichotomous scale (clear or unclear) and to provide suggestions as to how to rewrite the statements to make the language clearer. Any obscurity was discussed and resolved by the expert committee, and the final version of the Lithuanian CASP-19 was conducted.Study sample.The cross-sectional study was performed on ambulatory men and women aged 60 and older. Exclusion criteria were: mother language is not Lithuanian, Mini-Mental State Examination (MMSE) < 25 points, present acute illness, or malignant tumor. The elderly participants were recruited to the study by a convenience sampling method, after which interviews and meetings were scheduled with the researchers.Internal consistencyis an important measurement property for questionnaires that intend to measure a single underlying concept (construct) by using multiple items. It was evaluated by Cronbach’s alpha coefficient.Construct validity.In addition to the CASP-19 questionnaire, another QoL measure—EQ-5D—was also completed. The EQ-5D consisted of five dimensions titled mobility, self-care, usual activities, pain/discomfort, and anxiety/depression, and each dimension had three levels—no problems, moderate problems, or extreme problems. The patient indicated health state by ticking the box next to the most appropriate statement in each of the five dimensions. This decision resulted in a one-digit number that expressed the level selected for that dimension. The digits for the five dimensions were combined into a five-digit number that described the patient’s health state . ConstruTest-rest reliability.We received the second CASP-19 questionnaire from 34 participants, but after exclusion of two persons with more than 20% missing data and two persons whose health had changed between the administrations, we obtained a sample of 30 subjects for the evaluation of the test-retest reliability.Floor and ceiling effectswere defined as when a high percentage of the participants had the lowest or the highest score, respectively. If floor or ceiling effects were present, it meant that some items may have been missed in the lower or upper end of the scale, possibly limiting content validity.All subjects gave their informed consent for inclusion before they participated in the study. The study was conducted in accordance with the Declaration of Helsinki, and the protocol was approved by Vilnius Regional Biomedical Research Ethics Committee .A Shapiro–Wilk test was used to determine normality of quantitative variables. Variables were reported as mean ± standard deviation for normally distributed data, and as median with interquartile ranges (IQR) for non-normally distributed data. The presence of significant differences between the two groups (men and women) was calculated using Student’s t-test and Mann-Whitney’s U-test for quantitative variables without normal distribution. The questionnaire’s internal consistency and homogeneity was evaluated by Cronbach’s alpha coefficient. A value between 0.70–0.90 indicated a good internal consistency. A value lower than <0.70 was considered questionable, and >0.90 indicated there were redundancies in the questionnaire . Pearsonp < 0.05.The statistical analysis was performed using IBM SPSS Statistics 18.0 software. Results were considered statistically significant based on probability, The study sample consisted of 132 participants with an average age of 73.1 ± 8.1 years and was comprised of 28 (21.8%) men and 103 (78.2%) women. Most participants (72.7%) were living in an urban area. Analyzing education level, we found that 89 (59.8%) were well educated, 14 (10.6%) had finished vocational or professional school, 26 (19.7%) had a post-secondary or special secondary school education, and 13 (9.9%) had finished secondary school. Of the participants, 56 (42.4%) were widowed, 21 (15.9%) had never married, 19 (14.4%) were separated/divorced, and 36 (27.3%) were still married. Detailed scores from the CASP-19 and EQ-5D questionnaires for men and women are shown in According to the Lithuanian Statistics department database , the parShapiro-Wilk normality testing revealed that CASP-19 results were normally distributed. The potential range of CASP-19 was from 0, which represented “a complete absence of quality of life” to 57, which represented “total satisfaction of control, autonomy, self-realization and pleasure domains.” The range of scores in this study was 12–57. The scale exhibited a slightly negative kurtosis with a mean of 35.8, a median of 37, and a standard deviation of 10.2. No participant scored zero and only two participants achieved the highest possible score of 57; therefore, floor and ceiling effects were not significant.General Cronbach’s alpha coefficient was 0.852, indicating a high level of internal consistency. The inter-item correlations ranged from 0.065 to 0.56, with the weakest area observed for question A6, “Family responsibilities prevent me from doing what I want to do”. Among participants who completed a second CASP-19 questionnaire after an interval of two weeks, we found a good agreement level between test-retest measures with an ICC of 0.82 (95% CI 0.79–0.85).The correlation matrix of total scores and the four domains of CASP-19 was assessed using Pearson’s correlation coefficient .Correlation coefficients ranged from 0.39 to 0.84. The CASP-19 total score correlated strongly with each of the domains—control, autonomy, self-realization, and pleasure. The strongest correlation was between CASP-19 total score and the self-realization domain score, while the weakest was between autonomy and self-realization domain scores.Results of construct validity of CASP-19 when compared with EQ-5D questionnaires are shown in r = 0.39, p < 0.001), the scores of the EQ-5D mobility domain , and the EQ-5D usual activities domain . We found negative correlations between the CASP-19 total score and the EQ-5D self-care domain , the EQ-5D pain/discomfort domain , and the EQ-5D anxiety/depression domain . Based on these results, the CASP-19 questionnaire was a reliable and valid instrument which could be used for clinical practice and research purposes.Data of the EQ-5D questionnaire were not normally distributed; therefore, we used Spearman’s correlation method to evaluate correlations between the total score of CASP-19 and the individual domains scores of the EQ-5D questionnaire. We found moderate correlations between the CASP-19 total score and the scores of the EQ-5D index domain . Men scored significantly higher on CASP-19 than women in all countries: Czech Republic , Russia , and Poland . Polish men and women reported the highest CASP-19 scores. The lowest CASP-19 scores were reported for Russian men, a score similar to what we found with Lithuanian women. However, the total CASP-19 score of Lithuanian men was the lowest. This could be explained by the different life expectancies for men and women in Lithuania; in 2016, life expectancy was 69.49 years for men and 80 years for women [r = 0.66) correlation between CASP-19 and Satisfied With Life Scale (SWLS) scores, but lower correlations between CASP-19 and SF-12 physical and mental components (r = 0.53 and r = 0.49). According of Bowling and Stenner [r = 0.49) and the Office of National Statistics (ONS) national Omnibus (r = 0.74) survey data. Correlation between CASP-19 and World Health Organization Quality of Life (WHOQoL-OLD) assessment scores were almost the same in the Ethnibus survey (r = 0.38) and in the ONS Omnibus survey (r = 0.69). The HAPIEE study results showed correlations between CASP-19 and Center for Epidemiologic Studies Depression Scale (CES-D) scores—Czech Republic population (r = −0.49), Russian population (r = −0.40), and Polish population (r = −0.57). Correlation between CASP-19 and SF-10 scores were the same in Czech Republic and Russia (r = 0.40), and slightly higher in Poland (r = 0.41) [We have found that the CASP-19 mean scores were lower in men than women . According to results from the Health, Alcohol, and Psychosocial factors in Eastern Europe (HAPIEE) study , there wor women . The conor women  reported = 0.41) .The limitations of our study were that the CASP-19 questionnaire was tested only on citizens of Vilnius district and that the study sample was rather small. Results could be different when citizens of other regions of Lithuania are surveyed.The analysis of the psychometric properties of the Lithuanian version of the CASP-19 questionnaire indicates that it is reliable and valid. The Lithuanian version of the CASP-19 questionnaire may be used to evaluate quality of life in elderly people.


Aeromonas salmonicida infections, we isolated and characterized five lytic A. salmonicida phages, AS-szw, AS-yj, AS-zj, AS-sw, and AS-gz. The results of morphological and genomic analysis suggested that all these phages are affiliated to the T4virus genus of the Caudovirales order. Their heterogeneous lytic capacities against A. salmonicida strains were demonstrated by experiments. A series of phage cocktails were prepared and investigated in vitro. We observed that the cocktail combining AS-gz and AS-yj showed significantly higher antimicrobial activity than other cocktails and individual phages. Given the divergent genomes between the phages AS-yj and AS-gz, our results highlight that the heterogeneous mechanisms that phages use to infect their hosts likely lead to phage synergy in killing the host. Conclusively, our study described a strategy to develop an effective and promising phage cocktail as a therapeutic agent to combat A. salmonicida infections, and thereby to control the outbreak of relevant fish diseases. Our study suggests that in vitro investigations into phages are prerequisite to obtain satisfying phage cocktails prior to application in practice.As an alternative approach against multidrug-resistant bacterial infections, phages are now being increasingly investigated as effective therapeutic agents. Here, aiming to design an efficient phage cocktail against  Aeromonas salmonicida is a waterborne pathogenic bacterium considered to be one of the most relevant fish pathogens and is the causative agent of furunculosis in aquaculture systems worldwide  are natural viruses that specifically infect bacteria . To datePseudomonas aeruginosa associated chronic otitis, which conferred significant improvement in clinic. Vibrio spp.  and assessed their ability in inhibiting A. salmonicida growth in vitro. The results suggest that the most efficient combination consists of two phages. Our optimized phage cocktail was capable of inactivating the fish pathogen over a period of 80 h and displayed higher efficacy, broader temperature, and pH adaptation ranges against the pathogen than other phage cocktails designed in this study. Taken together, our study shows that the two-phage cocktail is a promising agent for the management of A. salmonicida infections in aquaculture.Here, we isolated five lytic bacteriophages infecting Table 1 and Supplementary Table A. salmonicida strains used in this study were isolated from the intestinal homogenates of various diseased fishes collected from different geographical locations in China. A. salmonicida (KC254648.1 and KC254649.1) were purchased from the State Key Laboratory of Freshwater Ecology and Biotechnology   were provided by the State Key Laboratory of Microbiology Resources . A. salmonicida (MF632072) was provided by Sichuan Agricultural University . A. hydrophila strains  were isolated from the wastewater of an aquaculture farm in Zhanjiang City, Guangdong Province. The rest of the strains were isolated from various environmental samples. All of the strains were stored at -80°C and fresh plated bacterial cultures were maintained for less than 3 days in solid LB medium at 4°C prior to conducting experiments (600 = 0.2–0.6). We collected water samples that putatively contain Aeromonas phages from various locations in Guangdong Province, China. With the purpose of developing phage antimicrobial agents against the pathogen of A. salmonicida occurring in aquaculture in South of China, we selected 30°C as the operating temperature in this study.All of the bacterial strains used in this study were verified by 16S rRNA gene sequencing or complete genome sequencing  . A. salmeriments . One isoA. salmonicida strains based on the methods reported by A. salmonicida strains used in study. RAPD-PCRs were carried out as previously described with primers AP5, OPB-7, and AP3, respectively  was used as a host to screen the phages. Phage isolation was carried out according to the method reported by g for 10 min to remove the solid impurities, and the supernatants were filtered through a membrane filter (0.22 μm) to remove bacterial debris. To enrich the phages, 100 ml aliquots of the filtrates were mixed with 50 ml of A. salmonicida (MF663675) cultures and grown at 30°C overnight. The resulting cultures were centrifuged at 10,000 × g for 10 min, and the supernatant was filtered using 0.22 μm to remove residual bacterial cells. Next, the filtrate containing enriched phages (0.1 ml) was mixed with 0.5 ml of the host cells (OD600 = 0.4–0.9) in LB medium and 5 ml of molten top soft nutrient agar (0.7%). The mixture was overlaid onto solidified base nutrient agar (1.5%). After incubation for 4–8 h at 30°C, clear phage plaques were picked from the plate. Phages were purified according to the standard methods  in SM buffer  were adsorbed onto carbon-coated copper grids, and then were negatively stained with 2% phosphotungstic acid (pH 7.0). After drying at room temperature, the grids were examined using TECNAI G2 F20 S-Twin transmission electron microscope .Purified phage particles from a highly concentrated suspension , and the extracted phage DNA was sequenced using the Illumina Hiseq 1500 sequencer platform . The filtered reads were assembled into large contigs using IDBA-ud-1.0.9  following the manufacturer’s protocol. The DNA samples were visualized on 0.7% agarose gels containing SYBRud-1.0.9  and CLC 1.0.9  were added to the cultures (10 ml) of A. salmonicida (MF663675) cells (OD600 = 0.5) at a multiplicity of infection (MOI) of 0.001, and the mixtures were incubated at 30°C for 8–12 min. Next, 100 μl of the mixture was collected at specific time points: 0, 1, 2, 3, 4, 5, 10, 20, and 30 min, and diluted immediately with 900 μl of cold LB medium. After centrifugation , the supernatants were titrated to determine the number of un-adsorbed phages.Phage adsorption curves were generated based on the method reported by A. salmonicida (MF663675) cells (OD600 = 0.4–0.5) was centrifuged at 8,000 × g for 5 min. The cell pellets were re-suspended in 300 μl of LB medium, then 600 μl of phage suspensions were added to yield an MOI of 0.01. After adsorption for 5 min at 30°C, the mixtures were centrifuged at 12,000 × g for 10 min to remove any un-adsorbed phage particles prior to re-suspending the samples in 6 ml of LB medium. The samples were incubated at 30°C, and aliquots were collected at 10 min intervals over a 90 min period (120 min for phage AS-sw). These aliquots were immediately diluted, and phage titers were determined using the double layer agar method . Each phage was used at the same concentration (106 PFU/ml), and the phage cocktails consisting of two, three, four, and five phages were combined and evaluated, respectively. The killing curves of the different phage cocktails against the host A. salmonicida (MF663675), with the MOI of 0.01, were determined. Briefly, an aliquot of a single phage or a phage cocktail was mixed with the host A. salmonicida (MF663675) (108 CFU/ml) at the predetermined MOI and then evaluated using a Growth Curve instrument  to monitor the OD600 changes. Data were collected at 5 min intervals for 80 h at 30°C. For each assay, three control samples were included: the bacterial control (BC) was inoculated with the host bacteria only, and the phage control (PC) and the phage cocktail control (CB) were inoculated with phage(s) only. Samples were mixed with the host bacteria and incubated under the same conditions. For all phage infection assays, the initial titer of each phage was previously determined using the double agar layer method  and for various phage cocktail preparations. The optimal MOI was selected based on the killing curves of each phage at different MOIs  against r method . Three i8 PFU/ml) were incubated at the indicated conditions for 45 and 120 min, respectively. Phage titers and bacterial inactivation before and after bacterial exposure to phages at the indicated conditions were determined using the double layer agar method . The existence of significant differences among the different conditions was assessed by Chi-square test. For each experimental set, the significance of the differences was assessed by Chi-square test. Three independent assays were carried out for each assay. A value of MF498773, MF498774, MF448340, MF498775, and MF479730, respectively.The complete genome sequences of the following phages have been submitted to GenBank: AS-szw, AS-yj, AS-zj, AS-sw, and AS-gz. Their accession numbers were A. salmonicida (MF663675) as the host: AS-szw, AS-yj, AS-zj, AS-sw, and AS-gz from the water samples in the south of China. The phages produced clear and round plaques with the diameters ranging from 0.38 to 1.2 mm, and the largest clear plaque was formed by AS-zj . The phages have icosahedric heads that are larger than 100 nm in diameter. AS-yj is the largest, with a 120 nm icosahedric head and a 130 nm contractile tail.Transmission electron microscopy analysis of the phages showed that all five are morphologically similar to the phages of the A. salmonicida (MF663675), and approximately 90% (2.7 × 104 PFU/ml out of 3.1 × 104 PFU/ml) of the phage particles adsorbed to A. salmonicida (MF663675) within the first 5 min . One-step growth curves determined in LB medium at 30°C  showed that they have different patterns of one-step curves, suggesting that they have distinct genotypes. Among the five phages, AS-yj has the shortest latency time (less than 20 min), followed by AS-zj (20 min), AS-gz (30 min), AS-szw (40 min), and AS-sw (50 min). The phage AS-szw has the highest burst size (145 PFU/host), followed by AS-gz (135 PFU/host), AS-yj (98 PFU/host), AS-sw (86 PFU/host), and AS-gz (86 PFU/host) .All the five phages showed strong adsorption to the surface of Aeromonas sp. strains , and AS-zj has lytic activity against A. hydrophila (KJ806398.1). None of the phages can infect other 29 strains from the genera other than Aeromonas. We further typed the A. salmonicida strains using the RAPD and ERIC PCR primers, and the result showed different patterns of the PCR products , streptomycin (YK and BG), penicillin (YK and BG), sulfamethoxazole (YK and BG), and gentamicin (YK) . Each of the five phages has linear double-stranded DNA. The GC content of the genomes ranges between 38 and 42%. The average genome size of AS-szw, AS-yj, AS-zj, and AS-sw is 230,046  bp and AS-gz has the smallest genome . In total, 1891 ORFs were predicted, with an average of 378 ORFs per phage genome. The predicted proteins corresponding to ORFs were compared to the nr database of NCBI. In total, 84% (1488/1860) of the ORFs shared amino acid similarity (25–100%) with the sequences deposited in GenBank. The ORFs encoding putative functional proteins were assigned to the following clusters : DNA replication , DNA metabolism , DNA packaging (large subunit terminase), structural proteins , host lysis , and others . None of the known lysogenic-related genes was found within their genomes; therefore, we assumed all five are lytic phages. Homology-based searches using the predicted protein sequences did not identify any genes predicted to encode toxins, virulence factors, or proteins associated with antibiotic resistance, implying that these phages can be used as antibiotic agents against A. salmonicida infections but further investigations into the safety of the phages by fish challenges are needed before application.The genomes of the five phages were sequenced and characterized . The similar virion dimensions and genome sizes between AS-gz and Aeromonas phage phiAS4 (HM452125.1) support their close relationship . Phylogenetic analysis was performed based on the large subunit terminases identified in the genomes of AS-szw, AS-yj, AS-zj, AS-sw, AS-gz, as well as other reported phages  of the Secunda5virus genus . They are all members of the Myoviridae family. AS-szw, AS-yj, AS-zj, and AS-sw are in one clade with Aeromonas phage CC2 (JX123262.1). CC2 was reported to efficiently infect several virulent strains of A. hydrophila from diseased fish  using a series of MOIs   and the killing curves were determined. In Figure 5, the OD600 increased after 2.5 h at a MOI of 10, implying that heavy phage loads likely accelerated the occurrence of bacterial resistance to the phage infection in a short period of time. Notably, the killing curves indicated that AS-gz was highly effective against the host at all MOIs tested in this study. As seen from the killing curves, the efficacy of the phage load at MOIs of 0.01 was more effective than those of other MOIs against the host, which is evident by the shortened latency time and the inhibited host growth for the remaining duration of the experiment after 6 h .Phage AS-gz was firstly selected to treat Figure 5), and can be theoretically overcome by employing phage cocktails composed of multiple phages. Based on the characters of the isolated phages , we designed a combinatorial series of phage cocktails and investigated their ability in controlling A. salmonicida cells at the optimal MOI of 0.01. The bacterial killing curves of the cocktails composed of AS-yj, AS-szw, AS-zj, or AS-sw appeared to have not more significant advantages in controlling bacteria growth as we can see from Figure 6 that their latency times were 10 h and the bacterial cells grew up to 0.6 of OD600 within 30 h. We observed high performance of the cocktails containing the phage AS-gz in bacteria growth inactivation studies. The bacterial cell density remained less than 0.2 (OD600) throughout the duration of the experiment (80 h), suggesting that AS-gz likely played a dominant role in bacterial growth control when it was part of the cocktails. Particularly, slight differences of the killing curves also can be observed among the cocktails containing AS-gz. Notably, the cocktail comprised of AS-yj and AS-gz maintained its bacterial inactivation with an OD600 < 0.05 for 80 h . Plaque counting showed that AS-yj and AS-gz maintained their lytic capacity at pH 5–10  and 4–11 , respectively. Interestingly, the cocktail displayed relatively greater stability in lytic capacity with its infectivity over 80% at pH 5–8  than either of the individual phages . It was observed that the high temperature treatment inactivated 66% (resulting in 6.57 × 107 PFU reduction) and 100% (108 PFU reduction) of AS-yj at 60 and 80°C, respectively . Phage AS-gz appeared to be more sensitive to high temperature, with approximately 90% (9.1 × 107 PFU reduction) decrease in titer observed after incubation for 15 min at 60°C, and it was completely inactivated at 80°C for 15 min . Although it seems that the cocktail, with a combination of AS-yj and AS-gz, has not more significant advantages than the individual phages as shown in Figure 7F, which was drawn according to phage particle counting, evaluation according to bacterial inactivation determined by the killing curves still revealed that the cocktail (AS-yj and AS-gz) displayed a stronger lytic ability than individual phages with OD600 less than 0.2  and 18°C  rapidly increased when infected at higher MOIs (10 and 1), although the bacteria cells were inactivated earlier than at lower MOIs (0.1 and 0.01). Comparing of the killing curves of the phages against the host at MOIs of 0.1 and 0.01 resulted in the optimal MOI as 0.01 that was used in next step experiments. This observation also suggests that heavy phage loads likely result in the emergence of bacterial resistance to the phage infection. As reported by Multiplicity of infection is an important parameter when phages are applied to challenge bacteria. In this study, it is of interest to observe that phage-resistant bacterial variants may be induced by heavy phage load more rapidly than those treated with low load. As shown in 600 at an early time points . Aiming to broaden host-range, Figure 6). These data imply too many types of phages present in a given cocktail will cause competitive inhibition of phage infection against the host. Also, the cocktail composed of AS-gz and AS-yj has several advantages, such as higher efficacy in inactivating their hosts, higher stability under different temperature, and pH than any of the individual phages. In particular, we noted that phage AS-gz has a genome sequence that is greatly different from those of the other four A. salmonicida phages, suggesting that AS-gz likely uses a different infection mechanism against the host from the others, highlighting the importance of the knowledge obtained from genome sequencing prior to phage cocktail preparation.In practice, it was inevitable to observe that phage-resistant variants rapidly emerge when a single phage type was used to control specific pathogens . In thisFigure 6). The rapid emergence of phage-resistant bacteria is the main concern for phage therapy. It has been known that bacteria have many mechanisms to evade phage infection  we used in this study] using the software CRISPRfinder online7 (data not shown). We did not find any CRISPR or CRISPR-like system in the genomes. Thus, we speculate here that other mechanisms that likely helped the A. salmonicida strains evade the phage infection, such as: (1) preventing phage adsorption, which includes blocking of phage receptors , the production of extracellular matrix , and the production of competitive inhibitors (Escherichia coil); (2) preventing phage DNA entry, which depends on different superinfection exclusion (Sie) systems ; (3) cutting phage nucleic acids, including restriction–modification (R–M) systems (Streptococcus pneumoniae) and the CRISPR-Cas system (P. aeruginosa); and (4) abortive infection (Abi) systems, which target the crucial step of phage multiplication including replication, transcription, or translation  for bio-control of A. salmonicida infections in aquaculture environments.We aimed to develop an alternative antimicrobial agent against multidrug-resistant LC and YM conceived and designed the experiments. LC and SY performed the experiments. LC, QL, and GM analyzed the data. JY and DD contributed sample materials. SY and YM wrote the paper. YM, CL, BZ, and CL managed the project.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.